Breast cancer is the most frequent womens' cancer. One third of new breast cancers are estrogen receptor a (ER) protein negative and have a worse prognosis than ER positive breast cancers. The ER is a ligand activated transcription factor. Estrogen:ER binding stimulates rapid Src activation that feeds back to phosphorylate ER and increases its transcriptional activity. Estrogen rapidly activates ubiquitin-dependent ER proteolysis which in turn regulates ER activity. Our data suggest that Src-stimulates ER proteolysis and this is linked to activation of certain ER target genes. Src induction increased both ER-activated gene expression and ER proteolysis. Src inhibition increased ER stability. The weakly ER +, MDA-MB-361 and ER negative, BT-20 breast cancer lines both have high Src, and while ER protein synthesis was easily detected, the ER t1/2 was reduced. ER was increased by both estrogen deprivation and proteasome inhibitors in both ER + and ER- line studied. SiRNA to Src increased ER protein in BT-20. E6AP is an ubiquitin ligase that also acts as an ER- coactivator. We show E6AP acts as ubiquitin ligase for ER in vitro and E6AP-mediated ER ubiquitylation was increased by ER pre-treatment with Src kinase. Src inhibitors impaired ubiquitylation and degradation of ER in vivo and in vitro. Src activity was increased in primary ER negative breast cancers compared to ER positive. We further investigate if Src, when recruited by activated ER, regulates transcription-coupled ER proteolysis. Our Hypothesis is that a subset of ER negative breast cancers are estrogen responsive: they express ER mRNA but ER protein is undetectable due to accelerated Src and E6AP-mediated ER proteolysis. Oncogenic Src activation may phosphorylate the ER or key co-regulators to activate both ER proteolysis and ER target gene transcription. This is pursued in Aims 1-3: 1. To further test how Src activation affects ER levels in primary human breast cancers and in breast cancer lines; 2. To test whether Src mediated phosphorylation of the ER or of a key co-activator can promote ER ubiquitylation by E6AP and ER degradation in vitro and in vivo; and 3. To assay if Src activation modulates ER transcriptional activity. Oncogenic Src promotes breast cancer proliferation and survival, and may also accelerate ER proteolysis in breast cancers. Elucidation of mechanisms underlying the ER negative status of breast cancer may indicate why they are so aggressive and yield new therapeutic targets for this treatment-resistant breast cancer type.